Compositions and Methods to Enhance the Immune System

ABSTRACT

The invention relates to the field of molecular medicine. In particular, it relates to compositions and methods to enhance the clearance of aberrant cells, e.g. cancer cells or virus-infected cells, by the host&#39;s immune system. Provided is a composition comprising (i) a therapeutic compound that can trigger a host&#39;s immune effector cells against an aberrant cell, such as a therapeutic antibody, and (ii) at least one agent capable of reducing or preventing inhibitory signal transduction initiated via SIRPalpha.

The invention relates to the field of molecular medicine. In particular,it relates to compositions and methods to enhance the clearance ofaberrant cells, e.g. cancer cells or virus-infected cells, by the host'simmune system. Among others, it provides an enhanced efficiency of thetreatment of human subjects with a therapeutic antibody, in particularlythrough an increase in antibody-dependent cell mediated cytotoxicity(ADCC).

The immune system defends the body against infection, disease andforeign substances. It is made up of many organs and cells. An antigenis a substance that causes the immune system to make a specificresponse, called the immune response. Viruses, bacteria, germs, andparasites contain substances that are not normally present in the bodyand thus cause an immune response. The immune response can lead todestruction of the antigen and anything it is part of or to which it isattached. Several different types of cells are involved in the immunesystem's response to an antigen. Among the cells are macrophages,granulocytes, dendritic cells, natural killer cells and lymphocytes.Among the lymphocytes cells are B cells (B lymphocytes), T cells (Tlymphocytes), Killer T and Helper T cells.

Cancer cells have substances on their outer surfaces that can act asantigens and thus “mark” the cells as different or abnormal. Viruses,bacteria, and parasites have components that are substantially differentfrom normal human cells because they are truly foreign to the body andare detected by the immune system. However, the differences betweencancer cells and normal human cells may be more difficult for the immunesystem to detect. Cancer immunotherapies, typically employing monoclonalantibodies, are designed to help the immune system to recognize cancercells and/or to strengthen the immune response to the cancer cells andthus destroy the cancer.

Various therapeutic strategies in human beings are based on the use oftherapeutic antibodies. This includes, for instance, the use oftherapeutic antibodies developed to deplete target cells, particularlydiseased cells such as virally-infected cells, tumor cells or otherpathogenic cells. Such antibodies are typically monoclonal antibodies,of IgG species, typically with human IgG1 or IgG3 Fc portion. Theseantibodies can be native or recombinant antibodies, humanized miceantibodies (i.e. comprising functional domains from various species,typically Fc portion of human or non human primate origin, and variableregion or complementary determining region (CDR) of mice origin).Alternatively, the monoclonal antibody can be fully human throughimmunization in human Ig locus transgenic mice or obtained through cDNAlibraries derived from human cells. A particular example of suchtherapeutic antibodies is rituximab (Mabthera™; Rituxana), which is achimeric anti-CD20 monoclonal antibody made with human γ1 and κ constantregions (therefore with human IgG1 Fc portion) linked to murine variabledomains conferring CD20 specificity. In the past few years, rituximabhas considerably modified the therapeutical strategy against Blymphoproliferative malignancies, particularly non-Hodgkin's lymphomas(NHL). Other examples of humanized IgG1 antibodies include alemtuzumab(Campath™, which is used in the treatment of B cell malignancies ortrastuzumab (Herceptin™), which is used in the treatment of breastcancer.

Therapeutic antibodies achieve their therapeutic effect through variousmechanisms. They can have direct effects in producing apoptosis orprogrammed cell death in e.g. tumor cells. They can block growth factorreceptors, effectively arresting proliferation of tumor cells.

Indirect effects include recruiting cells that have cytotoxicity, suchas monocytes and macrophages. This type of antibody-mediated cell killis called antibody-dependent cell mediated cytotoxicity (ADCC).Monoclonal antibodies can also bind complement, leading to direct celltoxicity, known as complement dependent cytotoxicity (CDC).

While therapeutic antibodies represent a novel specific and efficientapproach to human therapy, particularly for treatment of tumors, they donot always exhibit a strong efficacy. For instance, while rituximab,alone or in combination with chemotherapy was shown to be effective inthe treatment of both low-intermediate and high-grade NHL, 30% to 50% ofpatients with low grade NHL have no clinical response to rituximab. Ithas been suggested that the level of CD20 expression on lymphoma cells,the presence of high tumor burden at the time of treatment or low serumrituximab concentrations may explain the lack of efficacy of rituximabin some patients. Nevertheless, the actual causes of treatment failureremain largely unknown. There is therefore a need in the art forincreasing the efficiency of the therapeutic antibodies.

Also, given the numbers of antibodies that have been tested in cancerindications, one might have predicted that anticancer antibodies wouldcomprise the vast majority of agents on the list of FDA approved drugs.However, only 4 out of the 12 antibody therapeutics on this list aretargeted for cancer therapy, and this appears largely due to the lack ofpatient benefit. Interestingly, it is now becoming clear that one of themain reasons for this is that cancer cells (like their healthycounterparts) are relatively resistant to immune-mediated killingmechanisms. The mechanism for this apparent resistance of cancer cellsagainst host immunity has not been established.

A goal of the present invention is therefore to identify means andmethods to enhance immunity and immunotherapy against aberrant cells,for example cancer cells. In particular, it is a goal to enhance the invivo efficacy of a therapeutic compound that can trigger a host's immuneeffector cells against an aberrant cell.

Interestingly, the present inventors discovered an endogenous mechanismthat limits the killing of aberrant cells, e.g. cancer cells, by immuneeffector cells (see FIG. 1). This mechanism involves the molecularinteraction between CD47, which is present on the surface of essentiallyall cells within the body of the host including cancer cells, and SIRPα,which is specifically expressed on immune cells, in particular onmacrophages and granulocytes (Adams et al., 1998. J. Immunol.161:1853-18592). Importantly, blocking the interaction between CD47 andSIRPα with antagonistic antibodies against either of the two componentswas found to dramatically enhance the in vitro killing of cancer cellsin the presence of anti-cancer cell antibodies (FIG. 2). This effect wasconfirmed in a murine lung tumour model in SIRPα-mutant mice (FIG. 3).These data show that the binding of CD47 to SIRPα generates aninhibitory signal that suppresses ADCC by the immune system. Withoutwishing to be bound by theory, it is hypothesized that interference withthe inhibitory signal via SIRPα leads to an enhanced activity of immuneeffector cells against an aberrant cells, presumably via an increase ofthe ADCC mechanism.

One aspect of the invention therefore relates to a compositioncomprising (i) a therapeutic compound that can trigger a host's immuneeffector cells against an aberrant cell and (ii) at least one agentcapable of reducing or preventing inhibitory signal transductioninitiated via SIRPα. For example, an agent is used which is capable ofinhibiting the interaction between SIRPα and CD47, such that theinhibitory signal via the CD47-SIRPα interaction is reduced. A host is amammal, preferably a primate or rodent, more preferably a human subject.

The therapeutic compound is a therapeutic antibody, in particular anantibody that induces or promotes antibody-dependent cellularcytotoxicity (ADCC). As used herein, ADCC is meant to encompassantibody-dependent cellular phagocytosis (ADCP) as well. Saidtherapeutic antibody is capable of forming an immune complex. In oneembodiment, the therapeutic antibody has a human or non-human primateIgG Fc portion. Preferably, the therapeutic antibody is a monoclonalantibody or a functional fragment or a derivative thereof, morepreferably a humanized, human or chimeric antibody. Said fragment or aderivative thereof is preferably selected from a Fab fragment, a F(ab′)2fragment, a CDR and a scFv. In a particular embodiment, the therapeuticantibody is an FDA approved therapeutic antibody, such as rituximab,herceptin, trastuzumab, alemtuzumab, bevacizumab, cetuximab orpanitumumab. See for example Strome et al., Oncologist 2007; 12;1084-1095.

According to the invention, an agent capable of reducing or preventinginhibitory signal transduction initiated via SIRPα is used to partiallyor fully block the inhibitory signal via the CD47-SIRPα complex. Agentscapable of reducing or preventing inhibitory signal transductioninitiated via SIRPα, e.g. by inhibiting the interaction between SIRPαand CD47, are known in the art and further agents can be identifiedusing known techniques based on a read-out of downstream signallingevents. For example, the interaction of cell-associated CD47 with SIRPαexpressed on the surface of myeloid cells is known to cause SIRPαtyrosine phosphorylation and to promote the recruitment and/oractivation of the tyrosine phosphatases SHP-1 and SHP-2 as well as anumber of other signalling proteins to the cytoplasmic part of the SIRPαprotein (Oldenborg P A et al. (2000) Science 288:2051-4, Oshima et al.(2002) FEBS letters 519:1-7). These components, and in particular SHP-1,and perhaps also SHP-2, are known to mediate the negative effects ofSIRPα triggering with respect to various downstream effects, includingthe phagocytosis of antibody- or complement-coated red blood cells(Oldenborg PA et al. (2001) J Exp Med. 193:855-62), and are thereforeanticipated to also mediate the inhibitory regulation of ADCC.Therapeutic agents inhibiting the CD47-SIRPα interaction will likewisealso prevent the recruitment and/or activation of SHP-1, SHP-2 and/orsome of the other indicated signalling molecules. A substance to reduceor prevent inhibitory signal transduction initiated via human CD47-SIRPαinteractions during ADCC can be selected using a series of assays aimedto detect: i) reduction of CD47-SIRPα interactions in general (Liu etal. (2007) J Mol Biol. 365:680-93, Liu et al. (2004) J. Immunol.172:2578-85) (FIG. 4), ii) reduction of CD47-dependent tyrosinephosphorylation of SIRPα and/or SHP-1 recruitment to SIRPα and resultantactivation of the SHP-1 tyrosine phosphatase activity (Oldenborg P A etal. (2000) Science 288:2051-4), and/or iii) enhancement of ADCC usingtherapeutic antibodies (e.g. trastuzumab, rituximab) against tumor(Mimura K et al. (2007) Oncology. 72:172-80, Shimadoi S et al. (2007)Cancer Sci. 98:1368-72, Lefebvre M L et al. (2006) J Immunother.29:388-97) or other cells (e.g. red blood cells).

WO99/40940 discloses ligands of CD47 and agents binding to said ligands,such as CD47 antibodies and SIRPα, for the treatment of inflammatory,autoimmune and allergic diseases, graft rejection and/or chroniclymphocytic leukaemia.

It has been reported that ligation of SIRPα with specific antibody Fabfragments can suppress the production of inflammatory mediators bymacrophages (Van den Berg et al., J. Leukocyte Biol. 1999; pg. 16)

WO02/092784 is related to polynucleotides and polypeptides relating tothe modulation of SIRPα-CD47 interactions.

Armant et al. disclose that anti-CD47 monoclonal antibodies selectivelysuppress IL-12 release by monocytes (J. Exp. Med. Vol. 190, 1999, pg.1175-1181).

Van den Berg et al. report that activated macrophages are the majorcause of tissue damage during inflammation in the CNS. Three antibodieswere selected which bind to the rat SIRPα receptor (J. Neuroimmunology,Vol. 90, 1998, pg. 53).

US2003/0026803 discloses a method for the treatment of an autoimmunedisease with macrophage involvement, comprising administering an agentwhich inhibits the interaction between CD47 and SIRPα. Also describedtherein are methods for identifying such agents.

However, the use of a CD47/SIRPα inhibitory agent as disclosed herein,namely to enhance a host's immune effector cells, has not been disclosedor suggested in the art.

An “agent” or “antagonist”, as referred to herein, may be substantiallyany molecule or process which is capable of achieving the requiredfunction, namely of reducing or preventing the CD47/SIRPα inducedsuppression of the cytolytic and/or phagocytic response of immuneeffector cells (see FIG. 1). This function is suitably achieved byinhibiting or interfering with the CD47-SIRPα interaction. “Inhibiting”the CD47/SIRPα interaction means that the functional relationshipbetween the two molecules is altered such as to reduce or eliminate thekilling-suppressive effects on the macrophage by CD47. For example, thebiological interaction between SIRPα and CD47 may be reduced or altered,thereby preventing inhibitory signalling induced through SIRPα.Alternatively, the inhibitory signalling through SIRPα may be preventedwithout actually affecting the interaction with CD47.

Inhibitory molecules of a variety of types are known in the art, and canbe used as a basis for the design of agents in accordance with thepresent invention. One or more agents of the same or of a different type(e.g. small molecule and antibody) may be used. In one embodiment, acomposition comprises a proteinaceous substance capable of inhibitingthe interaction between SIRPα and CD47. For instance, it is a peptide,an antibody or antibody fragment. Peptides according to the presentinvention are usefully derived from SIRPα, CD47 or another polypeptideinvolved in the functional SIRPα-CD47 interaction. Preferably, thepeptides are derived from the N-terminal V-type immunoglobulin domainsin SIRPα or CD47 which are responsible for SIRPα-CD47 interaction.

Preferred agents are antibodies or antibody fragments, which may bereadily prepared and tested as described below, using techniques knownin the art. For example, a composition comprises as antagonist of theCD47/SIRPα-induced signalling an anti-CD47 antibody, an anti-SIRPαantibody, or a fragment thereof.

Suitable CD47/SIRPα inhibitory agents for use in the present inventioncan be selected using an (high throughput) in vitro assay involvingco-incubation of tumor cells and macrophages in the presence of atherapeutic antibody against the tumor cells and testing the efficacy ofcandidate agents e.g. a panel of monoclonal antibodies against eitherCD47 or SIRPα, to enhance antibody-dependent killing. See alsoExample 1. For example, phagocytosis and cytolysis of cultured humanbreast cancer cells by human monocyte-derived macrophages (MDM) ormyelomonocytic cell lines mediated by a therapeutic antibody can beestablished in the presence and absence of a candidate antagonist agent.Such assay systems known in the art. For example, purified monocytes canbe cultured with GM-CSF, M-CSF, or no cytokine for five or six days.Antibody dependent cellular phagocytosis (ADCP) and cytolysis (ADCC)assays can be performed with the MDM and HER-2/neu positive target cells(SK-BR-3). ADCP can be measured by two-color fluorescence flow cytometryusing PKH2 (green fluorescent dye) and phycoerythrin-conjugated (red)monoclonal antibodies (MoAb) against human CD14 and CD11b. ADCC cansuitably be measured with established radioactive 51-Cr release assays,or by a commercial non-radioactive LDH detection kit. However, othermethods may also be used.

As will be understood, the present invention is advantageously used toenhance the in vivo efficacy of a therapeutic compound that can triggera host's immune effector cells against any type of aberrant cell. Asused herein, “aberrant cell” refers to any diseased or otherwiseunwanted cell in a host organism.

In one embodiment, it is a cancer cell. For example, it is anon-Hodgkin's lymphoma cell, a breast cancer cell, a chronic lymphocyticleukaemia cell or a colorectal cancer cell.

Clearly, the blocking of CD47-SIRPα interactions by suitable antagonistsoffers great promise for enhancing antibody-mediated destruction ofcancer cells. Principally, the added value of resolving the limitationsof antibody therapy against cancer can occur at at least three distinctlevels:

-   -   1. By decreasing the threshold of cancer cell killing, the        dosing and/or frequency of antibody treatment can be lowered,        resulting in a significant reduction of costs. This is of        relevance, since the production of antibody therapeutics, which        are generally humanized recombinant proteins, is expensive.    -   2. The cure- and survival-rates can increase significantly by        increasing the overall effectiveness of antibody therapy.    -   3. Increasing ADCC can have a dramatic effect on the range of        antibody therapeutics that would be suitable for clinical        application. Many antibody therapeutics that would otherwise not        have beneficial effects, may in combination with CD47-SIRPα        antagonists prove to be effective. In fact, a number of the        antibody therapeutics that have thus far not demonstrated        sufficient activity in trials should perhaps be reconsidered.

One of the strengths of the concept of the present invention resides inits broad applicability. In principle, it can be expected to potentiatethe effects of any therapeutic antibody against cancer, in particularthose that exert their effects, at least in part, by ADCC. Furthermore,therapeutic antibodies which have not shown any ADCC component in theabsence of CD47-SIRPα interference, may be able to raise a beneficialADCC response upon blocking of CD47-SIRPα interactions. As indicatedbefore most of the FDA-approved therapeutic antibodies are of the humanIgG1 subclass, which can in principle be expected to be efficientinducers of ADCC. Thus, the present invention can be practiced incombination with the majority of therapeutic antibodies.

One embodiment of the invention relates to the use of an agent capableof reducing or preventing inhibitory signal transduction initiated viaSIRPα inhibiting the interaction between SIRPα and CD47, in thepreparation of a medicament for the treatment or prophylaxis of adisease or disorder that would benefit from enhanced phagocytosis bymacrophages. Exemplary diseases that would benefit from enhancedphagocytosis by macrophages include cancer, such as non-Hodgkin'slymphomal, breast cancer, chronic lymphocytic leukaemia or colorectalcancer.

In fact, the treatment or prophylaxis of any disease or disorder whereinaberrant or otherwise unwanted cells are involved can benefit from theuse of an inhibitory agent as disclosed herein. In one aspect, saiddisease is a viral infection, in particular in infection caused by amember of the family Poxviridae. As will be understood, an inhibitoryagent, or a combination of two or more different inhibitory agents, maybe used in the manufacture of a medicament in combination with a furthertherapeutic compound. In a preferred embodiment, said furthertherapeutic compound can trigger a host's immune effector cells againstan aberrant cell.

In one aspect, the invention relates to a method of increasing ADCC in asubject receiving anti-cancer treatment, said method comprisesadministering to said subject prior to, simultaneously, before or afterthe administration of an anti-cancer medicament an agent capable ofreducing or preventing inhibitory signal transduction initiated viaSIRPα in an amount of sufficient to increase ADCC. For example, saidanti-cancer medicament is a therapeutic antibody which inhibits theinteraction between SIRPα and CD47. The subject to be treated is forexample a patient suffering from non-Hodgkin's lymphomal, breast cancer,chronic lymphocytic leukaemia or colorectal cancer.

In a related aspect, there is provided a method of increasing ADCC in asubject receiving therapeutic antibody treatment, said method comprisesadministering to said subject prior to, simultaneously, before or afterthe administration of said therapeutic antibody an agent capable ofreducing or preventing inhibitory signal transduction initiated viaSIRPα in an amount of sufficient to increase ADCC.

Also, the invention provides a method of increasing the efficiency of atherapeutic antibody treatment in a subject, said method comprisesadministering to said subject prior to, simultaneously, before or afterthe administration of said therapeutic antibody an agent capable ofreducing or preventing inhibitory signal transduction initiated viaSIRPα.

In another embodiment, the invention provides the use of an agentcapable of reducing or preventing inhibitory signal transductioninitiated via SIRPα for the treatment or prophylaxis of a viralinfection. In general, any method that promotes the host immune systemto respond more efficiently to the virus is likely to increase itsnatural and acquired (e.g. by vaccination) immunity against the virus.In a specific aspect, the invention provides the use of an agent capableof inhibiting the interaction between SIRPα and CD47 for the manufactureof a medicament for the treatment or prophylaxis of disease caused bypox virus. Poxviruses (members of the family Poxviridae) can infect as afamily both vertebrate and invertebrate animals. The prototype ofpoxvirus family is vaccinia virus, which has been used as a successfulvaccine to eradicate smallpox virus. Vaccinia virus is also used as aneffective tool for foreign protein expression to elicite strong hostimmune response. The name of the family, Poxviridae, is a legacy of theoriginal grouping of viruses associated with diseases that produced poxsin the skin. Modern viral classification is based on the shape andmolecular features of viruses, and the smallpox virus remains as themost notable member of the family. The only other poxvirus known tospecifically infect humans is the molluscum contagiosum virus (MCV).Although the World Health Organization (WHO) declared the virusofficially eradicated in 1977, post Sep. 11, 2001 the American and UKgovernments have had increased concern over the use of smallpox or smallpox like disease, in bio-terrorism.

It has been established that poxviruses encode a homologue of CD47,termed viral CD47 (vCD47). By interacting with SIRPα on immune effectorcells, the present inventors hypothesize that vCD47, in addition toendogenous CD47, can provide negative signals that prevent killingand/or phagocytosis of poxvirus-infected cells. In one embodiment, theinvention thus provides a composition comprising (i) a therapeuticcompound that can trigger a host's immune effector cells against avirally-infected cell, such as a cell infected by the poxvirus, and (ii)at least one agent capable of reducing or preventing inhibitory signaltransduction initiated via SIRPα. Suitable therapeutic compounds includeviral vaccines, preferably a poxviral vaccine.

The invention accordingly also provides the use of an inhibitory agentto reduce or prevent inhibitory signal transduction initiated viaSIRPalpha, for instance induced by vCD47-SIRPα interaction, to (i)increase the natural host resistance to infection with poxviralpathogens, (ii) to enhance the efficacy of vaccination against poxviralpathogens) and/or to (iii) enhance the effectiveness of vaccination withpoxviral vectors, such as vaccinia.

LEGENDS TO THE FIGURES

FIG. 1. Model for the role of CD47 and SIRPα in limiting theantibody-dependent killing of tumor cells by the immune system andpotentiation of tumor cell destruction by blocking CD47-SIRPαinteractions. Antibodies directed against the tumor cells are recognizedby immune cell Fc-receptors and this induces tumor cell killing. Undernormal conditions (left panel) this antibody-induced killing is limitedby the interaction of CD47 on the tumor cells with SIRPα on the immunecell, which generates an intracellular signal that negatively regulatesthe killing response. By blocking the interaction between CD47 and SIRPα(right panel) the antibody-induced killing of tumor cells is enhancedbecause it is relieved from this limitation.

FIG. 2: Blocking CD47-SIRPα interactions with antagonistic monoclonalantibodies enhances CC52 antibody-dependent cellular phagocytosis of ratCC531 colon carcinoma cells by rat NR8383 macrophages. CC531 tumor cellsand NR8383 macrophages were incubated in culture plates with medium inthe absence or presence of CC52 antibody against CC531 cells and/orblocking monoclonal antibodies against either CD47 or SIRPα. After 1.5hours the percentage of ADCP was determined. For details, see Example 1.

FIG. 3: SIRPα-derived signals limit the killing of B16 tumor cells invivo. CD47-expressing B16 melanoma cells were injected into control(i.e. Wild type) mice or into mice lacking the SIRPα cytoplasmic tailthat mediates intracellular signaling in immune cells (i.e. SIRPα−/−).Groups of mice were treated every other day for 2 weeks with asuboptimal dose of therapeutic antibody TA99 directed against the gp75tumor antigen present on the B16 cells. One week afterwards the animalswere sacrificed and lung tumor load was quantified. Representativepictures (panel A) from the lungs of these mice show that there isessentially no tumor formation in the antibody-treated SIRPα−/− mutantmice as compared to the antibody-treated wild type mice, identifying anegative role of SIRPα signaling in tumor cell elimination in vivo. Eachpoint in the graph (panel B) represents evaluation of a single animal.*, p<0.005, students T-test.

FIG. 4: ADCC of human monocytes towards Jurkat acute T leukemia cells isenhanced by blocking CD47-SIRPα interactions. (A) Surface expression ofCD3 (using CLB-T3/4.2a mAb) and CD47 (using B6H12 mAb) and on Jurkatcells as evaluated by flow cytometry. (B) ADCC of human monocytestowards Jurkat cells after pre-incubation with mouse IgG_(2a) anti-CD3(20 μg/ml) and/or B6H12 (50 μg/ml) anti-CD47 F(ab′)₂. Saturation ofJurkat cells with anti-CD47 F(ab′)₂ was confirmed by parallel flowcytometric staining (data not shown). Note that virtually no killing isinduced by anti-CD3 in absence of CD47-SIRPα blocking, whereassubstantial levels of killing are evoked in the presence of anti-CD47F(ab′)₂. Values shown are means±SD (n=3) from a representativeexperiment out of three.

FIG. 5: Rituximab-mediated ADCC of human monocytes towards RajiBurkitt's B cell lymphoma cells is enhanced by blocking CD47-SIRPαinteractions. (A) Surface expression of CD20 (using anti CD20 Rituximab)and CD47 (using anti-CD47 B6H12 mAb) and on Raji cells as evaluated byflow cytometry. Red histogram represents control and blue histogramrepresents the Rituximab (upper histogram) or CD47 staining (lowerhistogram) (B) ADCC of human monocytes towards Raji cells afterpre-incubation with Rituximab (20 μg/ml) and/or B6H12 (10 μg/ml)anti-CD47. ADCC was performed at an effector:target ratio of 50:1. Notethat the Rituximab-mediated killing of Raji cells is significantlyenhanced by anti-CD47 mAb. Values shown are means±SD (n=3) from arepresentative experiment. * statistically significant difference,p<0.05.

The invention is exemplified by the following examples.

EXAMPLE 1 In vitro Evidence For a Role of CD47-SIRPα Interactions inADCC

In order to investigate the contribution of the CD47-SIRPα interactionduring ADCC of tumor cells by macrophages an assay was employed in whichCC531 rat colon carcinoma cells were incubated with CC52 antibody andrat NR8383 effector cell macrophages.

Materials and Methods

Rat CC531 colon carcinoma cells and NR8383 rat alveolar macrophages wereroutinely cultured in RPMI-1640 medium containing 10% fetal calf serum(FCS) (Gibco BRL) and antibiotics. CC531 were detached from the tissueflasks by scraping, washed in PBS, and labelled with 5 μM of DiI(Molecular Probes) for 15′ at RT. After washing 3.75×10⁵ CC531 cells,either preincubated or not for 15′ with 5 μg/ml anti-rat CD47 antibodyOX101, were incubated, in a round-bottomed 96-well tissue cultureplastic plate in 200 μl of HEPES-buffered RPMI-1640 containing 0.5% BSA,with 1.25×10⁵ NR8383 cells, either preincubated or not for 15′ with 5μg/ml anti-rat SIRPα antibody ED9 or its Fab′-fragments, in the presenceor absence of CC531-reactive mAb CC52 (1 μg/m1). After incubation for90′ at 37° C. the cells were washed and stained using the macrophagespecific biotinylated antibody ED3 (directed against rat sialoadhesin)and FITC-labelled streptavidin. ADCP (expressed as the % of NR8383having ingested DiI-labelled CC531 cells) was determined on a FACScanflow cytometer (Becton and Dickinson).

Results

In the absence of blocking antibodies against CD47 (OX101) or SIRPα(ED9) only very little antibody-dependent cellular phagocytosis isobserved, whereas in the presence of such antibodies CC531 are readilyphagocytosed (FIG. 2). This shows that interactions between CD47-SIRPαon respectively tumor cells and macrophage effector cells can negativelyregulate ADCC in vitro.

EXAMPLE 2 In vivo Evidence For a Role of SIRPα Signalling inAntibody-Dependent Tumor Cell Killing

In order to demonstrate that SIRPα provides signals that inhibit tumorcell killing in vivo we compared antibody-dependent tumor cell killingin wild type and SIRPα-mutant mice (Yamao (2002) J Biol Chem.277:39833-9) using an in vivo B16F10 mouse melanoma model (Bevaart L etal. (2006) Cancer Res. 66:1261-4). The SIRPα mutant mice lack thecomplete cytoplasmic tail, including the ITIM motifs that act as dockingsites for SHP-1 and SHP-2.

Materials and Methods

Young adult (7 weeks old) C57B1/6 wild type or SIRPα-mutant mice (Yamao(2002) J Biol Chem. 277:39833-9) were injected i.v. 1.5×10⁵ B16F10melanoma cells (in 100 μL saline; obtained from the National CancerInstitute (Frederick, Md.), in the absence or presence of therapeuticantibody TA99 (10 μg/mouse at day 0, 2, 4, 7, 9, and 11 after tumor cellinjection). After 21 days the animals were sacrificed and the number ofmetastases and tumor load in the lungs was determined as described(Bevaart L et al. (2006) Cancer Res. 66:1261-4).

Results

As can be seen in FIG. 3 there was a significantly lower level of tumordevelopment in the SIRPα-mutant mice as compared to wild type mice usingsuboptimal concentrations of therapeutic monoclonal antibody TA99. Theseresults demonstrate that SIRPα is a negative regulator of tumor cellkilling in vivo.

EXAMPLE 3

To provide further evidence for a negative role of CD47-SIRPαinteractions in tumor cell killing by myeloid cells, we established anADCC assay employing human CD47-expressing Jurkat T cell leukemic cells,opsonized with a murine IgG_(2a) anti-CD3 antibody (Van Lier R A et al.Eur J Immunol. 1987; 17:1599-1604) as a target (FIG. 4A), and humanSIRPα-expressing monocytes as effector cells, and used this to test theeffect of antibodies that block CD47-SIRPα interactions.

ADCC assay

Monocytes were isolated by magnetic cell sorting by using anti-CD14coated beads according to the manufacturer's instructions (MiltenyiBiotec B.V., Utrecht, The Netherlands) from PBMC isolated by densitycentrifugation using isotonic Percoll (Pharmacia Uppsala, Sweden) fromheparinized blood obtained from healthy volunteers. The cells werecultured for 16 h in complete RPMI supplemented with 5 ng/ml recombinanthuman GM-CSF (Pepro Tech Inc, USA), harvested by mild trypsin treatment,and washed. Jurkat cells (5-8×10⁶ cells) were collected and labeled with100 μCi ⁵¹Cr (Perkin-Elmer, USA) in 1 ml for 90 min at 37° C. Whereindicated the cells were preincubated with anti-CD47 and/or anti-CD3,and washed again. Monocytes were harvested and seeded in 9-well U-bottomtissue culture plates in RPMI with 10% FCS medium. The target cells(5×10³/well) and effector cells were co-cultured in 96-well U-bottomtissue culture plates in complete medium at a ratio of E:T=50:1 for 4hours at 37° C., 5% CO₂. Aliquots of supernatant were harvested andanalyzed for radioactivity in a gamma counter. The percent relativecytotoxicity was determined as [(experimental cpm-spontaneouscpm)/(Total cpm-spontaneous cpm)]×100%. All samples were tested intriplicate.

Results

As can be seen in FIG. 4B, anti-CD3-mediated ADCC against Jurkat cells,which express high levels of surface CD47 (FIG. 4A), is potently andsynergistically enhanced by saturating concentrations ofF(ab)′₂-fragments of the antibody B6H12 that blocks CD47 binding toSIRPα. Notably, in the absence of effector anti-CD3 antibody no effectof anti-CD47 F(ab)′₂ was observed, suggesting that CD47-SIRPαinteractions act selectively to restrict antibody- andFc-receptor-mediated affects on tumor cell killing.

Collectively, these data demonstrate that CD47-SIRPα interactions, andthe resultant intracellular signals generated via SIRPα in myeloidcells, form a barrier for antibody-mediated destruction of tumor cells.These results provide a rationale for employing antagonists of theCD47-SIRPα interaction in cancer patients, with the purpose of enhancingthe clinical efficacy of cancer therapeutic antibodies.

EXAMPLE 4 Blocking of CD47-SIRPα Interactions Enhances the Effect ofAnti-Cancer Therapeutic Antibodies

In order to demonstrate that the blocking of CD47-SIRPα interactionsindeed enhance the effect of established anti-cancer therapeuticantibodies, we developed an ADCC assay using human Raji Burkitt's Blymphoma cells as targets, human monocytes as effector cells, and anFDA-approved therapeutic antibody against CD20 (Rituximab). Forexperimental details see the legend to FIG. 5. As can be seen in FIG. 5,the blocking antibody against CD47, B6H12, was able to significantlyenhance the Rituximab-mediated cytotoxicity towards Raji cells.

1-18. (canceled)
 19. A method of enhancing antibody-dependent cellularcytotoxicity (ADCC) comprising contacting a target cell with (i) anantibody comprising a human or non-human primate IgG Fc portion, whereinthe antibody induces ADCC and (ii) an agent capable of reducing orpreventing inhibitory signal transduction initiated via SIRPα, whereinsaid contacting occurs in the presence of an effector cell.
 20. Themethod of claim 19, wherein the target cell is a diseased cell.
 21. Themethod of claim 20, wherein the diseased cell is a cancer cell.
 22. Themethod of claim 21, wherein the cancer cell is a non-Hodgkin's lymphomacell, a breast cancer cell, a chronic lymphocytic leukemia cell or acolorectal cancer cell.
 23. The method of claim 19, wherein the targetcell is a virally infected cell.
 24. The method of claim 19, wherein theantibody comprises a human IgG Fc portion.
 25. The method of claim 24,wherein the human IgG Fc portion is a human IgG1 Fc portion.
 26. Themethod of claim 24, wherein the human IgG Fc portion is a human IgG3 Fcportion.
 27. The method of claim 19, wherein the antibody comprises anon-human primate IgG Fc portion.
 28. The method of claim 19, whereinthe antibody is rituximab, herceptin, trastuzumab, alemtuzumab,bevacizumab, cetuximab or panitumumab.
 29. The method of claim 19,wherein the agent is an anti-SIRPα antibody.
 30. The method of claim 19,wherein the agent is an anti-SIRPα Fab, F(ab′)2, or ScFv.
 31. The methodof claim 19, wherein the agent is an anti-CD47 antibody.
 32. The methodof claim 19, wherein the agent is an anti-CD47 Fab, F(ab′)2, or ScFv.33. The method of claim 19, wherein the contacting occurs in vitro. 34.The method of claim 19, wherein the contacting occurs in a humansubject.
 35. The method of claim 19, wherein the effector cell is amonocyte.
 36. The method of claim 19, wherein the effector cell is amacrophage.